Marjorie A. Liénard

Allelic variation in a fatty-acyl reductase gene causes divergence in moth sex pheromones

Pheromone-based behaviours are crucial in animals from insects to mammals, and reproductive isolation is often based on pheromone differences. However, the genetic mechanisms by which pheromone signals change during the evolution of new species are largely unknown. In the sexual communication system of moths (Insecta: Lepidoptera), females emit a species-specific pheromone blend that attracts males over long distances. The European corn borer, Ostrinia nubilalis, consists of two sex pheromone races, Z and E, that use different ratios of the cis and trans isomers of acetate pheromone components. This subtle difference leads to strong reproductive isolation in the field between the two races, which could represent a first step in speciation. Female sex pheromone production and male behavioural response are under the control of different major genes, but the identity of these genes is unknown. Here we show that allelic variation in a fatty-acyl reductase gene essential for pheromone biosynthesis accounts for the phenotypic variation in female pheromone production, leading to race-specific signals. Both the cis and trans isomers of the pheromone precursors are produced by both races, but the precursors are differentially reduced to yield opposite ratios in the final pheromone blend as a result of the substrate specificity of the enzymes encoded by the Z and E alleles. This is the first functional characterization of a gene contributing to intraspecific behavioural reproductive isolation in moths, highlighting the importance of evolutionary diversification in a lepidopteran-specific family of reductases. Accumulation of substitutions in the coding region of a single biosynthetic enzyme can produce pheromone differences resulting in reproductive isolation, with speciation as a potential end result.

Evolution of multicomponent pheromone signals in small ermine moths involves a single fatty-acyl reductase gene

Fatty-acyl CoA reductases (FAR) convert fatty acids into fatty alcohols in pro- and eukaryotic organisms. In the Lepidoptera, members of the FAR gene family serve in the biosynthesis of sex pheromones involved in mate communication. We used a group of closely related species, the small ermine moths (Lepidoptera: Yponomeutidae) as a model to investigate the role of FARs in the biosynthesis of complex pheromone blends. Homology-based molecular cloning in three Yponomeuta species led to the identification of multiple putative FAR transcripts homologous to FAR genes from the Bombyx mori genome. The expression of one transcript was restricted to the female pheromone-gland tissue, suggesting a role in pheromone biosynthesis, and the encoded protein belonged to a recently identified Lepidoptera-specific pgFAR gene subfamily. The Yponomeuta evonymellus pgFAR mRNA was up-regulated in sexually mature females and exhibited a 24-h cyclic fluctuation pattern peaking in the pheromone production period. Heterologous expression confirmed that the Yponomeuta pgFAR orthologs in all three species investigated [Y. evonymellus (L.), Yponomeuta padellus (L.), and Yponomeuta rorellus (Hübner)] encode a functional FAR with a broad substrate range that efficiently promoted accumulation of primary alcohols in recombinant yeast supplied with a series of biologically relevant C14- or C16-acyl precursors. Taken together, our data evidence that a single alcohol-producing pgFAR played a critical function in the production of the multicomponent pheromones of yponomeutids and support the hypothesis of moth pheromone-biosynthetic FARs belonging to a FAR gene subfamily unique to Lepidoptera.

Key biosynthetic gene subfamily recruited for pheromone production prior to the extensive radiation of Lepidoptera

BackgroundMoths have evolved highly successful mating systems, relying on species-specific mixtures of sex pheromone components for long-distance mate communication. Acyl-CoA desaturases are key enzymes in the biosynthesis of these compounds and to a large extent they account for the great diversity of pheromone structures in Lepidoptera. A novel desaturase gene subfamily that displays Δ11 catalytic activities has been highlighted to account for most of the unique pheromone signatures of the taxonomically advanced ditrysian species. To assess the mechanisms driving pheromone evolution, information is needed about the signalling machinery of primitive moths. The currant shoot borer, Lampronia capitella, is the sole reported primitive non-ditrysian moth known to use unsaturated fatty-acid derivatives as sex-pheromone. By combining biochemical and molecular approaches we elucidated the biosynthesis paths of its main pheromone component, the (Z,Z)-9,11-tetradecadien-1-ol and bring new insights into the time point of the recruitment of the key Δ11-desaturase gene subfamily in moth pheromone biosynthesis.ResultsThe reconstructed evolutionary tree of desaturases evidenced two ditrysian-specific lineages (the Δ11 and Δ9 (18C>16C)) to have orthologs in the primitive moth L. capitella despite being absent in Diptera and other insect genomes. Four acyl-CoA desaturase cDNAs were isolated from the pheromone gland, three of which are related to Δ9-desaturases whereas the fourth cDNA clusters with Δ11-desaturases. We demonstrated that this transcript (Lca-KPVQ) exclusively accounts for both steps of desaturation involved in pheromone biosynthesis. This enzyme possesses a Z11-desaturase activity that allows transforming the palmitate precursor (C16:0) into (Z)-11-hexadecenoic acid and the (Z)-9-tetradecenoic acid into the conjugated intermediate (Z,Z)-9,11-tetradecadienoic acid.ConclusionThe involvement of a single Z11-desaturase in pheromone biosynthesis of a non-ditrysian moth species, supports that the duplication event leading to the origin of the Lepidoptera-specific Δ11-desaturase gene subfamily took place before radiation of ditrysian moths and their divergence from other heteroneuran lineages. Our findings uncover that this novel class of enzymes affords complex combinations of unique unsaturated fatty acyl-moieties of variable chain-lengths, regio- and stereo-specificities since early in moth history and contributes a notable innovation in the early evolution of moth-pheromones.

Functional consequences of sequence variation in the pheromone biosynthetic gene pgFAR for Ostrinia moths

Pheromones are central to the mating systems of a wide range of organisms, and reproductive isolation between closely related species is often achieved by subtle differences in pheromone composition. In insects and moths in particular, the use of structurally similar components in different blend ratios is usually sufficient to impede gene flow between taxa. To date, the genetic changes associated with variation and divergence in pheromone signals remain largely unknown. Using the emerging model system Ostrinia, we show the functional consequences of mutations in the protein-coding region of the pheromone biosynthetic fatty-acyl reductase gene pgFAR. Heterologous expression confirmed that pgFAR orthologs encode enzymes exhibiting different substrate specificities that are the direct consequences of extensive nonsynonymous substitutions. When taking natural ratios of pheromone precursors into account, our data reveal that pgFAR substrate preference provides a good explanation of how species-specific ratios of pheromone components are obtained among Ostrinia species. Moreover, our data indicate that positive selection may have promoted the observed accumulation of nonsynonymous amino acid substitutions. Site-directed mutagenesis experiments substantiate the idea that amino acid polymorphisms underlie subtle or drastic changes in pgFAR substrate preference. Altogether, this study identifies the reduction step as a potential source of variation in pheromone signals in the moth genus Ostrinia and suggests that selection acting on particular mutations provides a mechanism allowing pheromone reductases to evolve new functional properties that may contribute to variation in the composition of pheromone signals.

Semi-Selective Fatty Acyl Reductases from Four Heliothine Moths Influence the Specific Pheromone Composition

Background Sex pheromones are essential in moth mate communication. Information on pheromone biosynthetic genes and enzymes is needed to comprehend the mechanisms that contribute to specificity of pheromone signals. Most heliothine moths use sex pheromones with (Z)–11–hexadecenal as the major component in combination with minor fatty aldehydes and alcohols. In this study we focus on four closely related species, Heliothis virescens, Heliothis subflexa, Helicoverpa armigera and Helicoverpa assulta, which use (Z)–11–hexadecenal, (Z)–9–tetradecanal, and (Z)–9–hexadecenal in different ratios in their pheromone blend. The components are produced from saturated fatty acid precursors by desaturation, β–oxidation, reduction and oxidation. Results We analyzed the composition of fatty acyl pheromone precursors and correlated it to the pheromone composition. Next, we investigated whether the downstream fatty–acyl reduction step modulates the ratio of alcohol intermediates before the final oxidation step. By isolating and functionally characterizing the Fatty Acyl Reductase (pgFAR) from each species we found that the pgFARs were active on a broad set of C8 to C16 fatty acyl substrates including the key pheromone precursors, Z9–14, Z9–16 and Z11–16:acyls. When presenting the three precursors in equal ratios to yeast cultures expressing any of the four pgFARs, all reduced (Z)–9–tetradecenoate preferentially over (Z)–11–hexadecenoate, and the latter over (Z)–9–hexadecenoate. Finally, when manipulating the precursor ratios in vitro, we found that the pgFARs display small differences in the biochemical activity on various substrates. Conclusions We conclude that a pgFAR with broad specificity is involved in heliothine moth pheromone biosynthesis, functioning as a semi–selective funnel that produces species–specific alcohol product ratios depending on the fatty–acyl precursor ratio in the pheromone gland. This study further supports the key role of these in pheromone biosynthesis and emphasizes the interplay between the pheromone fatty acyl precursors and the Lepidoptera specific pgFARs in shaping the pheromone composition.

Sex Pheromone Evolution Is Associated with Differential Regulation of the Same Desaturase Gene in Two Genera of Leafroller Moths

Chemical signals are prevalent in sexual communication systems. Mate recognition has been extensively studied within the Lepidoptera, where the production and recognition of species-specific sex pheromone signals are typically the defining character. While the specific blend of compounds that makes up the sex pheromones of many species has been characterized, the molecular mechanisms underpinning the evolution of pheromone-based mate recognition systems remain largely unknown. We have focused on two sets of sibling species within the leafroller moth genera Ctenopseustis and Planotortrix that have rapidly evolved the use of distinct sex pheromone blends. The compounds within these blends differ almost exclusively in the relative position of double bonds that are introduced by desaturase enzymes. Of the six desaturase orthologs isolated from all four species, functional analyses in yeast and gene expression in pheromone glands implicate three in pheromone biosynthesis, two Δ9-desaturases, and a Δ10-desaturase, while the remaining three desaturases include a Δ6-desaturase, a terminal desaturase, and a non-functional desaturase. Comparative quantitative real-time PCR reveals that the Δ10-desaturase is differentially expressed in the pheromone glands of the two sets of sibling species, consistent with differences in the pheromone blend in both species pairs. In the pheromone glands of species that utilize (Z)-8-tetradecenyl acetate as sex pheromone component (Ctenopseustis obliquana and Planotortrix octo), the expression levels of the Δ10-desaturase are significantly higher than in the pheromone glands of their respective sibling species (C. herana and P. excessana). Our results demonstrate that interspecific sex pheromone differences are associated with differential regulation of the same desaturase gene in two genera of moths. We suggest that differential gene regulation among members of a multigene family may be an important mechanism of molecular innovation in sex pheromone evolution and speciation.

Elucidation of the sex-pheromone biosynthesis producing 5,7-dodecadienes in Dendrolimus punctatus (Lepidoptera: Lasiocampidae) reveals Delta11- and Delta9-desaturases with unusual catalytic properties

Sex pheromones produced by female moths of the Lasiocampidae family include conjugated 5,7-dodecadiene components with various oxygenated terminal groups. Here we describe the molecular cloning, heterologous expression and functional characterization of desaturases associated with the biosynthesis of these unusual chemicals. By homology-based PCR screening we characterized five cDNAs from the female moth pheromone gland that were related to other moth desaturases, and investigated their role in the production of the (Z)-5-dodecenol and (Z5,E7)-dodecadienol, major pheromone constituents of the pine caterpillar moth, Dendrolimus punctatus. Functional expression of two desaturase cDNAs belonging to the Delta 11-subfamily, Dpu-Delta 11(1)-APSQ and Dpu-Delta 11(2)-LPAE, showed that they catalysed the formation of unsaturated fatty acyls (UFAs) that can be chain-shortened by beta-oxidation and subsequently reduced to the alcohol components. A first (Z)-11-desaturation step is performed by Dpu-Delta 11(2)-LPAE on stearic acid that leads to (Z)-11-octadecenoic acyl, which is subsequently chain shortened to the (Z)-5-dodecenoic acyl precursor. The Dpu-Delta 11(1)-APSQ desaturase had the unusual property of producing Delta 8 mono-UFA of various chain lengths, but not when transformed yeast were grown in presence of (Z)-9-hexadecenoic acyl, in which case the biosynthetic intermediate (Z9,E11)-hexadecadienoic UFA was produced. In addition to a typical Z9 activity, a third transcript, Dpu-Delta 9-KPSE produced E9 mono-UFAs of various chain lengths. When provided with the (Z)-7-tetradecenoic acyl, it formed the (Z7,E9)-tetradecadienoic UFA, another biosynthetic intermediate that can be chain-shortened to (Z5,E7)-dodecadienoic acyl. Both Dpu-Delta 11(1)-APSQ and Dpu-Delta 9-KPSE thus exhibited desaturase activities consistent with the biosynthesis of the dienoic precursor. The combined action of three desaturases in generating a dienoic sex-pheromone component emphasizes the diversity and complexity of chemical reactions that can be catalysed by pheromone biosynthetic fatty-acyl-CoA desaturases in moths.

Functional flexibility as a prelude to signal diversity? Role of a fatty acyl reductase in moth pheromone evolution.

Sex pheromones are the hallmark of reproductive behavior in moths. Mature females perform the task of mate signaling and release bouquets of odors that attract conspecific males at long range. The pheromone chemistry follows a relatively minimal design but still the combinatorial action of a handful of specialized pheromone production enzymes has resulted in remarkably diverse sexual signals that subtly vary in structure and in number and ratio of components. In a recent article,1 we showed that a single reductase gene (pgFAR) enables the conversion of key biosynthetic fatty-acyl precursors into fatty alcohols, the immediate precursors of the multi-component pheromone in small ermine moths (Lepidoptera: Yponomeutidae). In the light of the widespread usage of multi-component pheromone blends across Lepidoptera, it is likely that the pgFAR biochemical flexibility is a regular feature of the moth pheromone machinery and polyvalent reductase genes are emerging as pivots to promote phenotypic transitions in moth mating signals. In addition, the small ermine moth pgFAR nevertheless contributes to regulating the ratio among components. Here we show that the pgFAR substrate specificity is actually counterbalancing the inherent chain-length preference of an upstream desaturase with ∆11-activity and that the enzymes together modulate the final blend ratio between the Z11-16:OH, Z11-14:OH and E11-14:OH compounds before the final acetylation.